Juvenile hormone biosynthesis in the cockroach, Diploptera punctata: the characterization of the biosynthetic pathway and the regulatory roles of allatostatins and NMDA receptor by Juan Huang A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Cell and Systems Biology University of Toronto © Copyright by Juan Huang (2015) Juvenile hormone biosynthesis in the cockroach, Diploptera punctata: the characterization of the biosynthetic pathway and the regulatory roles of allatostatins and NMDA receptor Juan Huang Doctor of Philosophy (2015), Department of Cell and Systems Biology, University of Toronto Abstract The juvenile hormones (JH) play essential roles in regulating growth, development, metamorphosis, ageing, caste differentiation and reproduction in insects. Diploptera punctata, the only truly viviparous cockroach is a well-known model system in the study of JH biosynthesis and its regulation. The physiology of this animal is characterized by very stable and high rates of JH biosynthesis and precise and predictable reproductive events that correlate well with rates of JH production. Many studies have been performed on D. punctata to determine the function of JH. However, the pathway of JH biosynthesis has not been identified. In addition, although many factors are known to regulate JH biosynthesis, the exact mechanisms remain unclear. The aim of my research was to elucidate the JH biosynthetic pathway in D. punctata and study the mechanisms by which allatostatin (AST) and N-methyl-D-aspartate (NMDA) receptor regulate JH production. I have (1) identified genes in the JH biosynthetic pathway, and determined their roles in JH biosynthesis; (2) investigated the mode of action of AST by determining the signaling pathway of AstR and the target of AST action; (3) determined the role of the NMDA receptor in JH biosynthesis using RNA interference and treatment with an NMDA receptor antagonist. To validate the application value of my research, AST analogs with high JH inhibitory activity were designed and their activities on JH biosynthesis were measured by in vitro and in vivo bioassays. ii Acknowledgements It has been four and a half years since I began my studies at the University of Toronto. I still remember the first day in Toronto. I was excited and nervous. Starting from the first day I went to the lab, I was surrounded by friendly faces and help. My supervisor Stephen S. Tobe and his wife Martha Tobe helped me to get used to the culture on another continent. Jinrui Zhang helped me to find a place to live. And all the documents were handled in one day with the help of Ekaterina F. Hult and Jane Linley. They successfully took away all my nervousness and made me feel that my life in Toronto would be exciting. Before I came to Canada, I barely spoke English. Language has been troublesome for me. I was worried that no one would like to talk to me because of my poor English. But again, people in my lab, Ekaterina F. Hult, Jinrui Zhang, Koichiro J. Yagi, Shirley H. Tiu, Elisabeth Marchal and Ilke van Hazel helped me to get over my trouble. They have been super patient, and always encouraged me to speak. My English was greatly improved with their help. In regard to research, there are many people to thank. First, my supervisor Stephen S. Tobe; he has an interesting way to train his students. To start my project, Steve asked me to read papers and find a project I am interested in, instead of assigning me one. It was difficult in the beginning, but when I look back now, I find it was great training. Now I am able to start and complete a project independently, thanks to him. In addition, Steve always provided great suggestions for my projects, and he always encouraged me to try new things. He taught me: never to be afraid of failure, because that is part of PhD training. I would never have finished my PhD without his support and supervision. iii And I would also like to thank Ekaterina F. Hult and Elisabeth Marchal for their great help during my PhD study. I was a chemist before I came to Toronto. I knew very little biology. Ekaterina F. Hult not only helped me to start my project, but also taught me many techniques in biology. Most importantly, she has an ability to make me feel good about myself. She encouraged me many times when I was frustrated with my failed experiments. Elisabeth Marchal is the most kind and sweet person I have ever met. She is always very thoughtful and nice, and she always came up with great ideas. We were a great team and worked on several projects together. I learned a lot from her, not only her knowledge in biology, but also her attitude to research and life. I would also like to acknowledge my other collaborators who contributed to my research projects: Jinrui Zhang, who taught me Radiochemical assay, and cockroach dissection; Koichiro J. Yagi, who helped me set up HPLC and gave suggestions for my projects; Prof Barbara Stay, who taught me cockroach dissection and provided suggestions for my projects; Prof Jozef Vanden Broeck, who provided the equipment and reagents to run the AstR functional assays; Sven Zels, who taught me the technique of receptor functional assays; Ilke van Hazel, who helped me with the cell culture and the expression of NMDA receptors. I would like to express my gratitude to my committee members: Belinda Chang, Ian Orchard, William G. Bendena, David Lovejoy and Les Buck. I thank them for their great suggestions for my projects and my thesis. I would like to thank all my friends in Toronto and in China. They brought so much happiness and joy to my life, and made my life in Toronto pleasant and colorful. Lastly, I would like to thank my parents and my brother for their support in the past four and a half years. iv Table of Contents Abstract ……………………………………………………………………………………….ii Acknowledgement …………………………………………………………………………….iii Table of Contents……………………………………………………………………………...v Table of Figures………………………………………………………………………………..vii Table of Tables…………………………………………………………………………………ix Abbreviations…………………………………………………………………………………..x Chapter 1: General Introduction 1.1 Juvenile hormones………………………………………………………………… 1 1.2 JH biosynthetic pathway…………………………………………………………...7 1.3 JH signaling pathway……………………………………………………………... 26 1.4 Diploptera punctata………………………………………………………………. 32 1.5 Regulation of JH titre……………………………………………………………... 38 1.6 Rational and objectives of my study……………………………………………... 49 1.7 References………………………………………………………………………….. 52 Chapter 2: Characterization of the Juvenile Hormone pathway in the viviparous cockroach, Diploptera punctata 2.1 Summary…………………………………………………………………………... 69 2.2 Introduction……………………………………………………………………….. 69 2.3 Materials and Methods…………………………………………………………… 73 2.4 Results…………………………………………………………………………….... 80 2.5 Discussion………………………………………………………………………….. 90 2.6 Supplementary data………………………………………………………………. 96 2.7 References………………………………………………………………………… 100 Chapter 3: Mode of action of allatostatins in the regulation of juvenile hormone biosynthesis in the cockroach, Diploptera punctata 3.1 Summary………………………………………………………………………….. 105 v 3.2 Introduction………………………………………………………………………. 105 3.3 Materials and Methods…………………………………………………………... 108 3.4 Results……………………………………………………………………………...114 3.5 Discussion………………………………………………………………………….126 3.6 Supplementary data……………………………………………………………….131 3.7 References………………………………………………………………………… 134 Chapter 4: Identification and characterization of the NMDA receptor and its role in regulating reproduction in the cockroach, Diploptera punctata 4.1 Summary………………………………………………………………………….. 138 4.2 Introduction………………………………………………………………………. 138 4.3 Materials and Methods…………………………………………………………... 141 4.4 Results……………………………………………………………………………...144 4.5 Discussion………………………………………………………………………….155 4.6 Supplementary data……………………………………………………………….160 4.7 References………………………………………………………………………… 166 Chapter 5: General discussion 5.1 Function of JH in reproduction...……………………………………………….. 169 5.2 Evolution of the JH biosynthetic pathway...……………………………………. 170 5.3 Regulation of JH biosynthesis….………………………………………………... 172 5.4 The value of my study in insect control…..……………………………………...175 5.5 Future perspective……………………………………………………………….. 176 5.6 References………………………………………………………………………….177 Chapter 6: Appendices……………………………………………………………………… 180 vi Table of Figures Figure 1.1 Structures of the JH homologues in insects 2 Figure 1.2 The JH biosynthetic pathway 8 Figure 1.3 Dynamics of JH metabolism throughout the life cycle of D. punctata 35 Figure 2.1 Scheme of JH biosynthetic pathway 71 Figure 2.2 Tissue specific expression of genes encoding JH biosynthetic enzymes 82 Figure 2.3 Developmental expression of genes encoding JH biosynthetic enzymes 84 during the first gonadotrophic cycle of D. punctata Figure 2.4 The effect of JH precursors on JH biosynthesis by CA from mated female 86 D. punctata Figure 2.5 Efficiency of HMGR-JHAMT RNAi-mediated knockdown and the effect 86 of silencing on the transcription of the other genes encoding enzymes in the JH biosynthetic pathway in day 4 mated female D. punctata. Figure 2.6 JH regulates ovarian development 88 Figure 2.7 Transverse sections of the basal oocytes from day 4 control and HMGR- 89 JHAMT dsRNA-treated animals. Figure 3.1 Relative expression levels of Dippu-AstR and Dippu-AST mRNA in tissues 115 of day 4 males and mated females. Figure 3.2 Relative expression levels of Dippu-AstR and Dippu-AST mRNA during the 116 first gonadotrophic cycle Figure 3.3 The effect of Dippu-AST dsRNA on